Chapter 3
Tools in Microbiology
Inoculation
Introducing a sample (the inoculum) into a container with a nutrient medium
The
medium contains appropriate nutrients that sustains the growth of microorganisms
Some
microbes have to be inoculated into a living organism
Isolation: Separating one
species from another
Obtaining Pure Culture
Cultures
composed of cells arising from a single cell - PURE CULTURES
Types of media
1.
Physical state
Liquid
Solid
(agar)
2.
Chemical composition
Synthetic
Nonsynthetic (complex)
3.
Functional type
General
purpose
Enrichment
Selective
Differential
Measures to be taken when working with
microbiological media
-
Needs to
be sterilized
-
Prevent
contamination
·
Synthetic media
-
Known chemical composition (NaNO3 3g/l; glucose
2g/l
)
·
Non-synthetic (complex) media
-
Contains chemically undefined components (Pepton,
beef extract..)
·
Enrichment medium supports the growth of a specific group of microorganisms
(Ex. N2-fixing)
·
Selective media - favor specific microorganisms and inhibits the others
(methylene blue inhibits the growth of Gram+ bacteria)
·
Differential media - contain
substances that permit detection of microorganisms with specific metabolic
activity
Incubation
Microbiological cultures are placed in
temperature-controlled chambers incubators
Temperature: 20-400C
Pathogenic:
370C
MICROSCOPE The Instrument
Microscopes are the instruments that magnify the
cell (object) to extent at which the cell details become visible
Leeuwenhoeks microscope had one lens
Robert Hooke invented the compound microscope -
multiple lenses
Microscope The basic principle
The
specimen is magnified with the objective lens (real image)
This
image is magnified by ocular lens (virtual image)
An
enlarged and inverted image is received by retina
Basic features of microscopy
Magnification
Resolution
Contrast
Magnification
Magnification is the result of light refraction
Mag =
Objective Power x Ocular Lens Power
Ex:
Objective lens = 10X
Ocular Lens Power = 10X
Mag = 10 x 10 = 100X
Use of immersion oil with high power objectives
Immersion oil has the same refractive
index as the glass. Refractive index is a measure of relative velocity at which
light passes through a material
Resolution
Resolution (resolving power) is the ability of a lens to distinguish two
adjacent points as two separate objects. In light microscopes resolution is 0.2
mm (limit - 2000X)
How does the resolution depend on the
wavelength?
Resolving distance =
Wavelength of light /2 x NA (numerical aperture)
The shorter the wavelength - the greater the
resolution
Contrast
Specimen must contrast with their background
This can be achieved by:
Changing the refractive index of specimen
Stain the specimen
Types of Microscopes
Light Microscopes
1. Bright field
2. Phase contrast
3. Fluorescent
4. Dark filed
5. Differential Interference
6.Confocal
Electron Microscope
1. Transmission
2. Scanning
Light Microscopy - Compound Microscope
Optical microscope parts:
Illuminator,
Condenser,
Objective lens
Ocular
lens (eyepiece)
Dark-Field Microscopes
Best for
observing pale objects
Only
those light rays scattered by specimen enter objective lens
Specimen
appears light against dark background
Increases contrast and enables observation of more details
Fluorescent Microscopy
Fluorescence is the ability of certain substances to absorb short wavelengths of
light and emit light at a longer wavelength
Immunofluorescence
Diagnostic procedure:
Antibody
produced against a specific bacterium
Conjugate antibody and fluorochrome
Treat
the unknown bacterium
If
suspected bacteria are indeed present they will bind the tagged antibodies
Ultraviolet (or near) light is used as a light source
Phase Microscopes
Provides
better contrast and more details in the cell.
The
light rays that hit the specimen travel a different path than the rays, which do
not hit the specimen
Differential Interference Microscopy
(Nomarsky)
Uses two beams of light
Higher resolution
3-D images
Confocal Microscopy
Uses fluorescent dyes and UV lasers to illuminate
the sample
An image is taken in a single plane that is not
thicker than 1.0 μm
Resolution increased by up to 40% because emitted
light passes through pinhole aperture
Computer constructed 3-D images
Electron Microscopy
Two types of electron microscopes:
Transmission (TEM)
Scanning (SEM)
Source of illumination is an electron beam
Advantage of using EM
Resolving distance = Wavelength of light :2
Wavelength of visible light= 4000A
Resolution (light microscopy): 2000 A (0.2 mm)
1 angstrom = 1.0 Χ 10-10 meters
1 mm = 10-6 meters
E.M.
uses an electron beam as a source of illumination (100 000 times shorter
wavelength than visible light)
Resolution (EM): 2 A
Magnification 10,000X to 100,000X
Transmission electron microscope (TEM)
Image formed by the electrons transmitted
through a specimen
A specimen is a thin section of material (fixed,
embedded, and sliced never alive)
TEM is used for objects smaller than 0.2 mm
Scanning electron microscope
Used to
study the surface of the cell / tissue
Image
formed by the electrons reflected from
the surface 3-D view
PREPARATION OF SPECIMENS FOR OPTICAL MICROSCOPES
Wet
mount (living) preparation
Unstained
Stained (methylene blue)
Heat
fixed smear
Thin film of material containing microorganisms
is spread over the surface of the slide
Air dried
Heat fix (kill and fix bacteria to the slide)
Staining microbial cells
1. Fresh living preparations
2. Fixed, stained smears
Fixed smears:
Simple stains
Differential stains
Gram Stain
Acid-Fast Stain
Special stains
Negative (Capsule) Stain
Flagellar Stain
Fluorescent Stains
Endospore Stain